In the recent past, light-microscopy methods have been developed with which, based on a sequential, stochastic localization of individual point objects (in particular, fluorescence molecules), it is possible to display image structures that are smaller than the diffraction-limited resolution limit of classic light microscopes. Such methods are described, for example, in WO 2006/127692 A2; DE 10 2006 021 317 B3; WO 2007/128434 A1, US 2009/0134342 A1; DE 10 2008 024 568 A1; “Sub-diffraction-limit imaging by stochastic optical reconstruction microscopy (STORM),” Nature Methods 3, 793-796 (2006), M. J. Rust, M. Bates, X. Zhuang; “Resolution of Lambda/10 in fluorescence microscopy using fast single molecule photo-switching,” Geisler C. et al, Appl. Phys. A, 88, 223-226 (2007). This new branch of microscopy is also referred to as “localization microscopy.” The methods applied are known in the literature, for example, under the designations PALM, FPALM, (F)STORM, PALMIRA, or GSDIM.
The new methods have in common the fact that the structures to be imaged are prepared with markers that possess two different states, namely a “bright” state and a “dark” state. For example, if fluorescent dyes are used as a marker, the bright state is then a fluorescence-capable state and the dark state is a non-fluorescence-capable state. In order for image structures to be imaged at a resolution that is smaller than the classic resolution limit of the imaging optical system, a small subset of the markers is then repeatedly prepared into the bright state. This subset is referred to hereinafter as an “active subset.” The active subset must be selected so that the average spacing between adjacent markers in the bright state is greater than the resolution limit of the imaging optical system. The luminance signals of the active subset are imaged onto a spatially resolving light detector, e.g. a CCD camera. A spot of light whose size is determined by the resolution limit of the imaging optical system is therefore acquired from each marker.
The result is that a plurality of individual raw-data images are acquired, in each of which a different active subset is imaged. In an image analysis process, the center points of the spots of light (representing the markers that are in the bright state) are then determined in each individual raw-data image. The center points of the spots of light ascertained from the individual raw-data images are then combined into one overall depiction. The high-resolution image produced by this overall depiction reflects the distribution of the markers. For a representative reproduction of the structure to be imaged, a sufficiently large number of signals must be detected. But because the number of markers in the particular active subset is limited by the minimum average spacing that must exist between two markers in the bright state, a very large number of individual raw-data images must be acquired in order to image the structure completely. The number of individual raw-data images is typically in a range from 10,000 to 100,000.
The time required for acquiring an individual raw-data image is limited at the low end by the maximum image acquisition rate of the imaging detector. This leads to comparatively long total acquisition times for a series of individual raw-data images that is necessary for the overall depiction. The total acquisition time can thus amount to as much as several hours.
Movement of the sample being imaged, relative to the image-producing optical system, can occur over this long total acquisition time. Because all the individual raw-data images must be combined, after center-point determination, in order to create a high-resolution overall image, any relative motion between the sample and the image-producing optical system that occurs during the acquisition of two successive individual raw-data images degrades the spatial resolution of the overall image. In many cases this relative motion derives from a systematic mechanical motion of the system (also referred to as “mechanical drift”) that is caused, for example, by thermal expansion or contraction, by mechanical stresses, or by a change in the consistency of lubricants that are used in the mechanical components.
The effects described above will be illustrated below with reference to a conventional inverted light microscope, as depicted in FIG. 1. The microscope according to FIG. 1 has a U-shaped stand 2 to whose limbs a sample retainer 4 is attached. Sample retainer 4 encompasses a sample stage 6 and a holder 8, arranged on sample stage 6, on which a sample carrier (not further depicted in FIG. 1) having a sample is fastened. Located below sample stage 6 is an objective turret 10 having multiple objectives 12 that can be pivoted selectably into an imaging beam path that passes through a through hole 14 embodied in sample stage 6. The imaged sample can be viewed through an eyepiece 16. Also located on stand 2 is a port 18 at which a detector, e.g. a CCD camera, can be connected.
In order to select the sample region to be imaged, holder 8 can be moved, together with the sample carrier fastened to it, laterally (i.e. perpendicularly to the imaging beam path) on sample stage 6. A mechanical adjusting apparatus 20, depicted entirely schematically in FIG. 1, is provided for this. One problem here is that adjusting apparatus 20 is usually not embodied in as drift-stable a manner as is necessary for acquisition of the above-described high-resolution overall image using localization microscopy. If a mechanical drift occurs in adjusting apparatus 20, it is transferred to holder 8, which ultimately results in a lateral relative motion between the sample and objective 12 arranged in the imaging beam path, and thus in drifting of the individual raw-data images assembled into the overall image.
This kind of image drift in the individual raw-data images is also caused by the attachment of objective turret 10 to the U-shaped stand 2. As a result of this conventional arrangement, for example, the image drift-relevant distance between the imaging objective 12 and the sample arranged on holder 8 is comparatively large, since the sample is coupled to objective 12 via sample holder 8, sample stage 6, the U-shaped stand 2, and objective turret 10. Because of this comparatively long distance, the microscope according to FIG. 1 is susceptible to thermal instabilities and mechanical stresses that “add up” over the distance. The comparatively complex mechanism of objective turret 10 is also susceptible to drift.
Reference is further made to US 2004/0051978 A1, DE 11 2005 000 017 B4, and DE 1 847 180 U regarding the existing art.